1. Field of the Invention
The present invention relates to a method for the preparation of biocompatible collagen-glycosaminoglycan composite materials by vapor phase crosslinking with aldehydes.
2. Description of the Prior Art
Composite materials made of collagen and glycosaminoglycan (GAG) have been shown to be highly useful for certain biochemical applications. For example, U.S. Pat. No. 4,060,081 (Yannas et al, 1977), the teachings of which are hereby incorporated by reference, discloses a multilayer membrane suitable for use as synthetic skin. The bottom layer, which is placed in contact with a woundbed, is a highly porous lattice comprising collagen that is crosslinked with GAG. This lattice provides a biophysical supporting structure in which cells can migrate and proliferate to heal the wound.
The typical procedures that have been used in the past to prepare collagen/GAG composite materials are described in U.S. application Ser. No. 30,183, filed on Apr. 16, 1979, now U.S. Pat. No. 4,280,954 and U.S. application Ser. No. 169,897, filed July 17, 1980, now abandoned; the teachings of both of these applications are herein incorporated by reference. Briefly, a preferred embodiment of these procedures comprises the following steps, in sequence:
1. Mechanically cutting and grinding a source of collagen into particulate form.
2. Soaking the particulate collagen in dilute acetic acid.
3. Homogenizing the solution in a blender.
4. Adding a source of glycosaminoglycan which has been ground into particulate form. Typically, enough GAG is added to the solution to comprise about 6% to about 12% by dry weight of the composite material. The collagen/GAG mixture normally co-precipitates out of the acidic solution and forms a fibrous dispersion.
5. Homogenizing the precipitate in a blender.
6. Freezing the solution quickly in a shallow pan.
7. Subjecting the frozen dispersion to a high degree of vacuum, thereby causing the acidic fluid to evaporate while the spatial configuration of the partially crosslinked fibrils is maintained.
8. Contacting the freeze-dried product with a solution containing a crosslinking agent such as glutaraldehyde.
The composite material thus formed may be treated by additional procedures to remove all traces of aldehyde and to increase the crosslinking density and strength of the composite material.
Furthermore, U.S. Pat. No. 4,350,629 to Yannas et al discloses a method for improving the biocompatibility of the composite materials made by this general method. An aqueous dispersion of collagen is swollen in acid and contacted with a crosslinking agent prior to adding GAG to the solution. The resulting composite material causes extremely low or undetectable levels of blood platelet aggregation. The teachings of U.S. Pat. No. 4,350,629 are also hereby incorporated by reference.
Although the composite materials produced by the methods described in these patents and patent applications are extremely useful as synthetic skin and other prosthetic devices, certain problems still remain relating to storage and shelf-life of these materials.
In the current process for crosslinking porous sheets based on collagen and GAG, the sheets are placed in an aqueous solution of glutaraldehyde or a similar crosslinking agent. The crosslinked sheets are then rinsed in water and typically stored in an alcohol/water solution until ready for use as grafts. Storage in an alcohol/water solution has two disadvantages. First, the shelf-life of the collagen-GAG sheets in alcohol/water is limited to several weeks due to gradual degradation of collagen in the medium. Second, storage in a liquid makes transporation of the artificial skin cumbersome.
Both of these problems could be resolved if the collagen-GAG sheets could be crosslinked and stored in a dry state. Such an alternative procedure would allow storage of the sheets in the dry state with attendant increased shelf-life and significantly increase convenience in transporation. Unfortunately, freeze drying of the porous sheets following wet processing leads to significant loss in the porosity of the sheets and in substantial or total loss of performance of the sheets as grafts for the treatment of wounds. A different treatment is therefore needed for the production of artificial skin. Ruderman et al, J. Biomed. Mater. Res., 7, 263-265 (1973), discloses a vapor-phase crosslinking of collagen sponges with formaldehyde. However, the tensile strength of the untreated sponge was approximately 5 times greater than the tensile strength of the treated sponges; i.e., the bonds formed by the reaction with formaldehyde were said to be weaker than the hydrogen bonds they replaced. Accordingly, this reference teaches against the use of vapor-phase treatment with aldehydes to produce crosslinking in synthetic skins since the strength of a synthetic skin is very important.
Accordingly, there remained prior to this invention a need for new methods for crosslinking collagen-GAG composite materials intended for use as artificial skin.